Minimal residual disease of CLL in our everyday hematological practice M. Doubek, J. Chovancová, B. Tichý, O. Stehlíková, H. Francová Skuhrová, M. Klabusay, K. Malinová, M. Krejčí, Y. Brychtová, Š. Pospíšilová, J. Mayer
Is MRD negativity the goal of CLL treatment in everyday practice? Moreton et al. Eradication of Minimal Residual Disease in B-Cell Chronic Lymphocytic Leukemia After Alemtuzumab Therapy Is Associated With Prolonged Survival. J. Clin. Oncol. 2005
Is MRD negativity the goal of CLL treatment in everyday practice? Contra Some studies demonstrated significantly prolonged OS in MRD- patients (Moreton et al., 2005; Del Poeta et al. 2005; Tam et al. 2008; Bosh et al. 2008) Clinical trial data vs. everyday practice should not be comparble CLL is disease of elderly (CIRS score) CLL maintenace toxicity CLL is incurable by conventional therapy CLL relapses are not immediately treated
Which CLL patient is suitable for MRD evaluation in our practice? Clinical trial Allo Tx (First line intensive therapy in younger patients)?
Which method is suitable for MRD evaluation in everyday practice? Flow cytometry RQ-ASO IgH PCR
MRD analysis RQ-ASO IgH PCR probe probe qpcr MRD monitoring in CLL allele (patient) specific primers, V H family specific LNA probe at DNA level, sensitivity at least 10-4
MRD analysis flow cytometry Standardized flow cytometric analysis of MRD in CLL Rawstron Protocol Clinical trial (our clinical trials); all patients after allo Tx Three antigen combinations CD5/CD19 and CD20/CD38 CD5/CD19 and CD81/CD22 CD5/CD19 and CD79b/CD43 Correlation with RQ-ASO IgH-PCR in CLL patients underwenting allo Tx
Flow cytometer BD FACSCanto II
Flow cytometry gating strategy active CLL 1. step: gate of CD19 + SS low from Leu 2. step: CD19 + SS low FS low CLL after therapy
Detection of CLL cells using CD20/CD38 and CD5/CD19 protocol CLL cells: low expression of both CD20 and CD38
Detection of CLL Cells Using CD22/CD81 and CD5/CD19 Protocol CLL cells: low expression of both CD22 and CD81
Detection of CLL Cells Using CD79b/CD43 and CD5/CD19 Protocol CLL cells: low expression of CD79b, dim expression of CD43 (CD43 positive)
Active CLL MRD - MRD + ( 20/38/5/19 (Protocol
MRD our pilot analysis The aim of our work was to compare detection of MRD in CLL patients using multiparametric flow cytometry and molecular biology approaches with detection of IgVH specific clones (RQ-ASO IgH-PCR).
MRD our pilot analysis Patients and Methods 17 CLL patients underwenting allogeneic Tx for refractory/relapsed disease (FLAMSA-RIC conditioning, n=15; Cy+TBI conditioning, n=2) MRD evaluated bi-monthly after allo Tx 397 samples analyzed
MRD our pilot analysis Results MRD positivity detected repeatedly after allo Tx in 12 patients In 9 patients MRD positivity detected after previous negativity In 3 patients persisent MRD positivity
MRD our pilot analysis Results Samples positive by PCR: 66.7% Samples positive by flow: 58.7% PCR negative, flow positive 1.3% PCR positive, flow negative 9.3%
MRD our pilot analysis Results Median time from molecular relapse to hematologic relapse: 17 months (6-38); n=4
flow [%] PCR vs. flow R=0.94 45 40 35 30 25 20 15 10 5 0-5 y = 4E-05x - 0,2003 R 2 = 0,8854 0 100000 200000 300000 400000 500000 600000 700000 800000 900000 PCR [kopie/10**6 GE]
22.1.2010 6.7.2009 18.12.2008 1.6.2008 14.11.2007 28.4.2007 10.10.2006 24.3.2006 5.9.2005 MRD case report I FT, male, 1961 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 transplantace 16.11.2005 PB MRD - flow % PB chimérismus % PB kopie/10x6 GE MRD - PCR Klouzavý průměr/2 (PB kopie/10x6 GE MRD - PCR) Klouzavý průměr/2 (PB MRD - flow %) Klouzavý průměr/2 (PB chimérismus %) stop imunosuprese CT 7.9.2006 negativní 8.3.2007 CT negativní 5.12.2007 CT Infiltrát 74 mm 14.5.2009 1. relaps po HSCT 14.5.2005 CT tumorózní masa v retroperitoneu 2.6.2009 3 cykly MP+R 14.7., 11.8., a 15.9.2009 4000 3500 3000 2500 2000 1500 1000 500 0
14.10.2009 6.7.2009 28.3.2009 18.12.2008 9.9.2008 1.6.2008 22.2.2008 14.11.2007 6.8.2007 28.4.2007 18.1.2007 MRD case report II JP, female, 1946 PB MRD - flow % chimérismus % PB kopie/10x6 GE MRD - PCR Klouzavý průměr/2 (PB kopie/10x6 GE MRD - PCR) Klouzavý průměr/2 (PB MRD - flow %) Klouzavý průměr/2 (chimérismus %) 60.00 50.00 40.00 30.00 20.00 10.00 0.00 transplantace 1.8.2007 stop MMF 4.9.2007 stop CsA 22.1.2008 podání DLI 13.3.2009 1. relaps po HSCT 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0
Conclusions Disadvantage of flow cytometry consisted in limited sensitivity in contrast to ASO-PCR, where the sensitivity reaches to 1 cell in 10 6. On the other hand, disadvantage of PCR remain in high costs and labor intensity of this technique. For this reason flow cytometry is technique of first choice in our practice. It ensures rapid sample processing and the sensitivity of detection reaches up to one tumor cell in 10 4-5 leukocytes.
University Hospital Brno and University Campus